Members of the genus Rickettsia are the etiologic agents of rocky mountain and other spotted fevers and endemic, scrub, and epidemic typhus, diseases that pose a health threat worldwide. Although public health measures, the availability of antibiotics, and improvements in health care have reduced the classical epidemic manifestations of these diseases, rickettsial disease remains a serious public health problem. Cases of epidemic typhus, a disease usually associated with war and famine, continue to occur even in the absence of these precipitating factors. In the United States, an animal reservoir (the flying squirrel) of the causative agent of epidemic typhus, Rickettsia prowazekii, has been identified and associated with cases of human illness. Unfortunately, safe and efficacious vaccines are not available for any of the rickettsial diseases. R. prowazekii is an obligate, intracellular parasitic bacterium. Unlike other intracellular bacteria, rickettsiae grow directly within the cytoplasm of their host rather than within phagosomes or phagolysozomes. The overall goal of this research is to increase our understanding of how this novel pathogen survives within human cells. Rickettsial genes will continue to be isolated using recombinant DNA techniques in order to elucidate rickettsial gene structure, function, and regulation and to provide rickettsial proteins in quantities suitable for characterization. Genes critical to rickettsial survival, such as those coding for enzymes of the tricarboxyllic acid cycle are primary targets. Direct cloning techniques, using the complementation of Escherichia coli mutants deficient in the individual enzymes, as well as the identification and isolation of rickettsial genes using specific gene probes will be employed. In order to analyze the expression of rickettsial genes, cloned genes or synthetic oligonucleotides will be used as probes to follow the synthesis of specific messenger RNA during growth of the rickettsiae within their host cells. In addition, experiments designed to define the limits of rickettsial genetic manipulation will be initiated. Areas to be examined include transformation of rickettsiae, rickettsial DNA restriction systems, and DNA recombination in the rickettsiae.